Targeting the glutamine-arginine-proline metabolism axis in cancer

Abstract Metabolic abnormalities are an important feature of tumours. The glutamine-arginine-proline axis is an important node of cancer metabolism and plays a major role in amino acid metabolism. This axis also acts as a scaffold for the synthesis of other nonessential amino acids and essential metabolites. In this paper, we briefly review (1) the glutamine addiction exhibited by tumour cells with accelerated glutamine transport and metabolism; (2) the methods regulating extracellular glutamine entry, intracellular glutamine synthesis and the fate of intracellular glutamine; (3) the glutamine, proline and arginine metabolic pathways and their interaction; and (4) the research progress in tumour therapy targeting the glutamine-arginine-proline metabolic system, with a focus on summarising the therapeutic research progress of strategies targeting of one of the key enzymes of this metabolic system, P5CS (ALDH18A1). This review provides a new basis for treatments targeting the metabolic characteristics of tumours.


Glutamine addiction of cancer cells
Metabolic abnormalities are an important feature of cancers 1 .Rapidly proliferating cancer cells rely mainly on glutamine for survival and proliferation 2 .Glutamine is a nutrient that supports energy production and the synthesis of cellular biomass 3 .Additionally, this nutrient contributes to the supply of both carbon and nitrogen 3 .Glutamine is a widely recognised nutrient that is employed by cancer cells to increase proliferation and promote survival under conditions of metabolic stress 4 .As a result, glutamine serves as a rate-limiting factor for cell cycle progression, and in certain cellular contexts, the cell cycle cannot reach the S phase under conditions of glutamine depletion 5 .
Researchers have obtained a deeper understanding of the reprogramming of glutamine metabolism in cancer cells 6 .Glutamine provides energy for nucleotide, amino acid and lipid biosynthesis in growing cancer cells through the tricarboxylic acid (tcA) cycle and via its nitrogen and carbon skeleton 2 .As a result, most cancer cells are glutamine dependent and unable to survive without exogenous glutamine, a condition known as "glutamine addiction" 4 .Harry eagle first demonstrated that the cervical cancer cell line Hela requires 10 to 100 times more glutamine than other amino acids to maintain growth 7 .A variety of human cancer cells (such as pancreatic cancer, glioblastoma and small cell lung cancer cells) are sensitivity to glutamine starvation, which interferes with the metabolism of cancer cells and consequently leads to growth arrest and death 8 .Gliomas are glutamine-addicted cells.increased expression of the glutamine transport protein SNAt3 in glioblastoma tissues has been reported to be a hallmark of malignant gliomas compared to low-grade gliomas and normal brain tissues 9 .Additionally, the glutamine transporter protein Slc1A5 (ASct2) is also overexpressed in human glioblastoma cell lines and rat c6 glioma cells 10 .c-Myc in gliomas determines glutamine addiction through the activation of genes that encode proteins necessary for glutamine metabolism and uptake, such as Slc1A5 and glutaminase (GlS) 8,11 .Studies have identified molecular mechanisms linking upregulated GlS expression, glutaminolysis activation and the oncogene c-Myc in human prostate cancer cells and Burkitt's lymphoma 12 .Hassanein et al. demonstrated the overexpression of Slc1A5 in adenocarcinoma, squamous cell carcinoma and neuroendocrine lung tumours 13 .takeuchi et al. showed that Slc7A5 is expressed in non-small cell lung cancer (NSclc) and that 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BcH) inhibits Slc7A5 to reduce the viability of lung cancer cells 14 .in marked contrast to normal cells, lung cancer cells depend on glutamine due to the abnormal activation of oncogenes and the absence of tumour suppressors.these changes not only increase glutamine import via the upregulation expression of glutamine transporters but also promote the expression of metabolic enzymes involved in the metabolism of glutamine.For instance, a frequently disrupted route in lung cancer is the expansion of MYc functions via translocations and amplifications 15 .Abnormal glutamine metabolism was found in the serum metabolomic profile of natural killer t-cell lymphoma (NKtcl) patients, and Slc1A1 was found to be a key regulator of altered glutaminolysis.ectopic expression of Slc1A1 accelerated cell proliferation and tumour development in vitro and in vivo by increasing cellular glutamine absorption, improving glutathione metabolic flux, and inducing glutamine addiction 16 .Glutamine addiction is a hallmark of clear cell renal cell carcinoma (ccRcc).Fu et al. revealed that ccRcc tumour cells with high cell-intrinsic glutamine metabolism activity had a worse survival rate than those with low activity.cell-intrinsic glutamine metabolism in ccRcc tumours can enhance the immunosuppressive function of treg cells through interleukin (il)-23 while inhibiting the cytotoxicity of cD8 t cells.these findings support the use of il-23 blockade as an effective therapeutic approach for treating glutamine-addicted ccRcc 17 .
However, there is significant variability in the response of cancer cell lines to glutamine deprivation, suggesting that not all cancer cells require exogenous glutamine for survival 18 .in breast cancer, basal-type cells rely on glutamine, while luminal-type cells tend to not rely on glutamine 19 .this difference in phenotype is linked to the luminal-specific expression of glutamine synthetase (GS), which is in turn regulated by a key luminal transcription factor called GAtA3.Furthermore, GS inhibits the expression of GlS to shift the metabolic pathway towards glutamine synthesis in luminal breast cells and increase the possibility of glutamine symbiosis with basal breast cells 19 .Kim et al. discovered that tumour GlS expression is greater in tNBc than in other subtypes of breast cancer, and GlS is usually negative in HR-positive tumours 20 .A prior investigation indicated that glutamine is synthesised by glutamine synthetase in luminal-type breast cancer but not in basal-type breast cancer.therefore, basal-type breast cancer or tNBc demonstrates elevated levels of GlS expression, rendering it more dependent on glutamine than other subtypes of breast cancer 18,19 .Furthermore, research has demonstrated that NSclc cell lines exhibit differing degrees of reliance on glutamine, with 15 out of 38 lines displaying dependence on glutamine based on a reduction in growth rate of 70% or more in glutamine-free medium.Another 15 cell lines demonstrated an intermediate dependence on glutamine, with a reduction in growth rate ranging from 30 to 70%.Finally, 8 out of 38 lines demonstrated relative insensitivity to glutamine deficiency, with a 30% or lower decrease in cell growth 21 .B lymphoblastoid cell lines, including promyelocytic Hl-60 cells, are highly reliant on glutamine for growth, while t-cell lines are able to proliferate in glutamine-free media.this dependence on glutamine is inversely related to the activity of GlS.Furthermore, GlS can be stimulated in glutamine-deficient media, especially glutamineindependent cells 22 .

Sources of glutamine
in human blood, glutamine accounts for 20% of the total amino acids, whereas in muscle, glutamine accounts for 60% of all amino acids, and the muscle is an important site for endogenous glutamine production.the exogenous glutamine and muscle protein ingested through the digestive tract are under the action of GlS, and the endogenous glutamine produced is the main source of human glutamine 23 .cells can acquire glutamine via two methods.One is the transport of glutamine into cells from the microenvironment via Slc transporters, including members of the Slc1, Slc6, Slc7, Slc36, and Slc38 families 24 .Both the Na + /amino acid exchanger Slc1A5 and the unidirectional Na + /cl -/amino acid isotropic transporter protein Slc6A14 are regulated by MYc and are often overexpressed in various types of cancer 24 .Furthermore, Slc7A5 (lAt1), which functions as a bidirectional transporter, facilitates the intracellular efflux of glutamine and the influx of leucine.Studies have shown that Slc1A5 mediates the entrance of glutamine into cells, which subsequently exits the cell due to the action of Slc7A5.this process is coupled with the entry of leucine into the cell, resulting in the synergistic activation of mammalian target of rapamycin (mtOR) signalling and thereby promoting cell growth and proliferation 25 .Notably, Slc3A2 is necessary for the formation of the Slc7A5-Slc7A11 heterodimer at the plasma membrane, which is also a target of MYc. in contrast, Slc6A14 is distinct from Slc1A5 and Slc7A5 because it transports substrates unidirectionally into cells, carries all essential amino acids, such as glutamine, and can couple Slc7A5 and Slc7A11 26 .
in the other method, cells synthesise glutamine through their own GS 27 .Mammals can synthesise glutamine in most tissues, and most free glutamine is synthesised and stored in skeletal muscle. in its physiological state, glutamine is generated by the combination of glutamate and ammonia through a reaction catalysed by GS. intracellular glutamate can be synthesised in two ways: (1) glutamate can be synthesised from α-KG by glutamate dehydrogenase or multiple aminotransferases, and (2) glutamate can be synthesised from other amino acids (e.g.proline, arginine, and histidine) 3 .Most likely due to excessive glutamine uptake by cancer cells, the glutamine concentration in the microenvironment of cancer tissues is significantly lower than that in other tissues 28 .thus, in glutamine-addicted cancer cells, some intrinsic oncogenic signalling pathways allow cancer cells to grow under conditions with a limited supply of glutamine 28 .

Destination of glutamine
the functions of glutamine are as follows: (1) Glutamine acts as a nitrogen donor for purines and pyrimidines, thus providing amide nitrogen to allow for de novo synthesis of nucleotides, amino sugars, and NAD cofactors 28 .(2) Glutamine acts as a nitrogen donor for nonessential amino acids, and glutamine can be converted to proline and ornithine 28 .(3) Glutamine acts as a carbon donor.endogenous and exogenous lipids are used by cells to form lipid membranes that are required for cell division.in xenograft models, inhibition of fatty acid synthesis prevents the growth of tumours, thus highlighting the importance of de novo lipid synthesis in tumour development.when hypoxia or mitochondrial dysfunction occurs, glutamine directly provides carbons for the synthesis of fatty acids and citrate 29,30 .
Glutamine is transported intracellularly via the Slc1A5 transporter and can also be transported back to the extracellular space after being exchanged with leucine by the lAt1 (heterodimer of Slc7A5 and Slc3A2) anti-transporter 31 .the first step in glutaminolysis is the generation of glutamate through a reaction catalysed by cytoplasmic or mitochondrial glutaminases (GlS1 or GlS2).Glutamate has several fates.First, glutamate can be directly converted to GSH by the glutamate-cysteine ligase catalytic (Gclc) subunit 28 .Second, glutamate can be converted to α-KG by GlUD (GlUD1 or GlUD2) or transaminase and enter the tricarboxylic acid cycle 32 .third, glutamate cyclisation produces proline.Glutamate is produced as γ-glutamyl phosphate (γ-GP) by the action of γ-glutamyl kinase (γ-GK) and later as γ-glutamyl semialdehyde (GSA) by the action of γ-GP reductase (γ-GPR).GSA is spontaneously cyclized to pyrroline-5-carboxylic acid (P5c), and P5c is reduced to proline by the action of pyrroline-5-carboxylic acid reductase (P5cR).Among these enzymes, γ-GK and γ-GPR are independent enzymes in bacteria and lower eukaryotes, whereas in higher organisms (e.g.humans and plants), γ-GK and γ-GPR are fused as single polypeptides to form a bifunctional enzyme, P5c synthase (P5cS), in which the γ-GK structural domain is the N-terminus and the γ-GPR structural domain is the c-terminus 33 .Fourth, glutamate generates arginine.in fact, glutamine, glutamate, proline and arginine are metabolic systems 34 .P5c generates ornithine under the action of ornithine delta transaminase (OAt), and ornithine then enters the urea cycle to generate arginine 35 .Remarkably, mammalian P5cS generates two isoforms, P5cS.short and P5cS.long, by selective splicing.P5cS.short is highly active in the intestine and is a key step in the catalytic arginine synthesis pathway, which can be inhibited by ornithine (Figure 1).P5cS.long is expressed in several types of tissues, is involved in glutamate-to-proline synthesis and is insensitive to ornithine inhibition (Figure 1); thus, this isoform allows cells to maintain proline synthesis when the ornithine levels are abnormally high 36 .Fifth, another major fate of glutamate derived from glutamine is extracellular excretion, in which glutamate can be exchanged with cystine via the xct (isomer of Slc7A11 and Slc3A2) reverse transporter and then transported back to the extracellular space 31 .

Glutamine metabolism
High levels of glutamine in the blood provide an easily accessible supply of carbon and nitrogen for biosynthesis, energy, and cellular homeostasis; cancer cells may use glutamine to accelerate the growth of tumours 31 .Glutamine is assumed to play a pleiotropic role in cellular activities 31 .Glutamine may be directly incorporated into proteins to regulate protein trafficking and translation.For the production of nonessential amino acids and nucleotides, glutamine delivers carbon and nitrogen through catabolic reactions 37 .thus, glutamine deficiency slows the development of some malignancies and may even cause cell death 38 .Additionally, some cancer cell lines use glutamine as a key nutrition source for oxidative metabolism 38 .Glutamine metabolism plays a crucial role in tumour progression by supporting mitochondrial oxidative phosphorylation, producing metabolic intermediates for the tcA cycle and for glutathione and NeAA synthesis and generating NADPH 39 .Recent studies have shown that pancreatic cancer cells use glutamine as a key fuel source for mitochondrial oxygen consumption.Additionally, the quantities of metabolites produced by glucose metabolism are impacted by variations in the expression of Slc1A5.intriguingly, this study on increased glutamine metabolism in cancer cells also demonstrated that glutaminolysis might support metabolic reprogramming, which suggests that glutamine metabolism is essential for carcinogenesis and tumour growth 40 .
the excessive proliferative of cancer cells requires a steady supply of fuels such as glucose and glutamine 38 .consequently, cancer cells regulate their metabolic pathways to meet their high nutrient demand.Dysregulated oncogenes are closely correlate with metabolic reprogramming, which increases glutamine consumption in cancer cells.Studies have attempted to elucidate the mechanism through which oncogenes alter metabolic pathways that promote cancer cell growth 41 .Notably, glutamine is essential for the survival of cancer cells with oncogenic MYc, K-Ras, and PiK3cA, and these cells use glutamine extensively for anabolic processes 42 .MYc is the oncogene that has most frequently been linked to increased glutamine metabolism 43 .MYc promotes the activity of the glutamine transporter Slc1A5, increases the expression of GlS, and activates the tcA cycle and glutathione synthesis under hypoxia 44 . in addition to mediating interactions with HeR2 and the eR in breast cancer, MYc may also rewire glutamine metabolism once other oncogenic pathways are activated 31 .through the overexpression of Slc1A5, glutamine absorption is increased in K-Rasdriven cells, similar to the results observed in MYc-driven cancer cells 45 .low glutamine levels cause cell death, whereas K-Ras knockdown protects cells from this apoptosis.K-Ras sensitises cells to glutamine deprivation 46 .taken together, these findings suggest that K-Ras-driven cancer cells exhibit increased glutamine metabolism and dysregulated cell growth, which promotes uncontrolled cell growth and facilitates glutamine acquisition and utilisation for cell growth 40 .

Proline metabolism
Proline, a nonessential amino acid and the only imino acid that can be synthesised by the body, is produced in the cytoplasm and can be converted into a variety of amino acids and metabolic intermediates.Proline is thus indirectly involved in the tricarboxylic acid and the urea cycles.Proline can be converted to glutamate by proline dehydrogenase (PRODH)/POX and P5cDH on the inner mitochondrial membrane, whereas glutamate is converted to proline by P5cS on the inner mitochondrial membrane and P5cDH in the cytoplasm.P5cS in the inner mitochondrial membrane and P5cR in the cytoplasm are used to convert glutamate to proline.Among these enzymes, P5cS is the key enzyme involved in the synthesis of proline from glutamate in organisms.in addition, P5c acts as a carbon skeleton and is an important intermediate metabolite in the interconversion of glutamate and ornithine that links glutamate metabolism and the ornithine metabolism cycle 47 .Ornithine can also be converted to proline through the action of ornithine transaminase and P5cR 48 .the metabolic relationship between glutamate and proline mediated by P5cS in cancer cells is shown in Figure 2.
Proline, an important amino acid metabolite, is involved in a variety of cellular signalling pathways and AtP energy metabolism processes 49 ; thus, proline affects many pathophysiological processes, such as cellular redox status; carbon and nitrogen metabolism; oxidative stress; and cell signalling, adaptation, and apoptosis, and plays an important role in tumorigenesis and progression 34 .Studies have shown that increasing proline level can increase the production of proteins, which are needed for cell proliferation.MYc and Pi3K signalling, which promotes cell proliferation, increases the gene expression of P5cS and PYcR 50 .increased expression of P5cS and PYcR1 is a poor prognostic factor for different types of cancers, which suggests that cancer cells require proline biosynthesis 51 . in addition, high expression of PYcR1 was detected in invasive breast ductal carcinoma, NSclc and prostate cancer cells.Knockdown of PYcR1 inhibits cancer cell growth 52 .Richardson et al. reported that de novo proline synthesis is the major metabolic change in metastatic breast carcinoma cells 53 .Similarly, Denkert et al. reported a significant increase in the proline levels in invasive ovarian cancer tumours compared with that in ovarian junctional tumours 54 .De novo proline synthesis is also significantly increased in melanoma cells compared with melanocytes 55 .vermeersch et al. demonstrated that the levels of metabolites in the proline and arginine metabolic pathways are significantly greater in ovarian cancer OvcAR-3 cells than in ovarian cancer stem cells.Similar to the finding that increased glycolytic content is an important carcinogenic phenotype, the reversal of proline accumulation in ovarian cancer stem cells may play a role in the stem cell-like phenotype 56,57 .Proline and ornithine (mainly proline) can induce the dose-and time-dependent differentiation of mouse embryonic stem cells (meScs) into a new epiblast stem cell (epiSc)-like state, and this state is induced by the breakdown of proline to P5c 58 .Proline is also considered a signalling molecule that controls stem cell behaviour, and physiological concentrations of proline are sufficient to convert eScs into mesenchymal-like, highly motile, and invasive pluripotent stem cells, which then can induce in vivo metastasis 59 .

Arginine metabolism
Arginine has multiple metabolic fates: arginine can be interchanged with glutamate and proline and serves as a precursor for the synthesis of proteins, nitric oxide, creatine, polyamines, agmatine, and urea 60 .Arginine is a conditionally essential amino acid that can be synthesised by the body itself in adult mammals under normal conditions, but under conditions such as starvation, inflammation, and small intestine or kidney dysfunction, the body does not synthesise a sufficient amount of arginine to meet metabolic requirements.therefore, in adult mammals, arginine is a semiessential amino acid, whereas in newborns or preterm infants, this amino acid is essential 61 .
there are three main sources of free arginine in organisms: dietary protein, endogenous synthesis, and protein turnover 62 .Studies have shown that approximately 50% and 40% of the carbon for endogenous arginine synthesis originates from intestinal glutamine and proline, respectively 63 .P5cS is the first step in the de novo synthesis of arginine.in the small intestine, P5c is converted to ornithine via OAt and then to citrulline via Otc.Subsequently, citrulline is converted to arginine in the kidney via the urea cycle cytoplasmic enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASl). in addition, under starvation conditions, a portion of the ornithine synthesised by intestinal P5cS and OAt is transferred to the liver to replenish the urea cycle 64 .Studies have shown that in the neonatal gut, most glutamine-derived P5c is converted to ornithine rather than proline, and ornithine is subsequently used for the efficient synthesis of citrulline and arginine 65 .
in contrast to the finding that a single enzyme synthesises arginine, five enzymes use arginine as a substrate 66 .(1) Arginyl-tRNA synthetase (ArgRS) uses arginine as a substrate but is not considered a metabolic enzyme, and charged tRNAArg is not only necessary for protein synthesis but also plays a role in protein degradation 61 .(2) Arginine decarboxylase (ADc) catalyses the conversion of arginine to cO 2 and agmatine 67 .(3) Arginine-glycine amidinotransferase (AGAt) is involved in the first step in creatine synthesis 68 .(4) there are two ARG isozymes.Arginine can be hydrolysed by cytoplasmic ARG1 and mitochondrial ARG2 to produce urea and ornithine, which in turn can serve as precursors for the synthesis of polyamines, proline, or glutamate 69 .(5) Nitric oxide synthases (NOSs) catalyse the oxidation of arginine to NO and citrulline (Figure 2) 69 .
ASS1 is recognised as the rate-limiting enzyme in arginine synthesis, and in many tumours, including hepatocellular carcinoma, melanoma, prostate cancer, and renal cancer, ASS1 expression is downregulated or even absent, which prevents these tumours from synthesising arginine and thus makes them dependent on exogenous arginine.these tumours are thus sensitive to arginine deprivation therapies and are termed arginine-nutrient-deficient tumours 70 .Arginine deprivation is emerging as a promising new clinical treatment strategy for metabolism-based cancer therapy 71 . in tumour cells that do not express ASS1, promoting the degradation of extracellular arginine triggers apoptosis 72 .Arginine deficiency leads to GcN2-dependent cycle arrest in Hcc cells, and inhibition of GcN2 in arginine-deficient Hcc cells enhances the efficacy of senescent compounds and promotes cellular senescence 73 .Arginine promotes the transition of activated t cells from glycolysis to oxidative phosphorylation.Additionally, arginine promotes the growth of more viable central memory-like cells that exhibit antitumour effects in mouse models 74 .Abnormal upregulation of ARG1 and ARG2 expression has been observed in a range of cancers.ARG1 overexpression increases eRK and AKt phosphorylation in neuroblastoma (NB), which promotes cell growth 75 .ARG2 expression is markedly greater in malignant thyroid tumours than in normal tissues, and the suppression of ARG2 decreases AKt expression while increasing tumour cell apoptosis 76 .Phosphorylated StAt3 can directly bind to the ARG1 promoter region of myeloid-derived suppressor cells (MDScs) in head and neck squamous cell carcinoma (HNScc) to stimulate transcription and enhance the immunosuppressive properties of MDScs 77 .ARG2 stimulates the metastasis of tumours in melanoma via the H2O2-StAt3 pathway 78 .

Interactions among glutamine-arginine-proline metabolism
Glutamine, arginine, and proline form a metabolic system with P5c as the central metabolite 34 .the interconnected networks of this metabolic system are shown in Figure 2. Proline is converted to arginine via the P5c intermediate.Arginase (ARG) then catalyses the forward conversion of arginine to ornithine, and OAt reversibly converts ornithine to P5c 79 .we were intrigued by the peculiar aspect of amino acid metabolism in which P5c is not only the precursor for proline synthesis but also the direct result of proline catabolism 34 .As a result, P5cS is the first enzyme that converts glutamate to proline 49 , and the glutamate-P5cS pathway accounts for most proline production in the human body 80 .Notably, proline cannot be metabolised by common amino acid enzymes (i.e.racemases, transaminases, and decarboxylases) because it contains an amino group in the pyrrolidine ring.instead, a specific family of enzymes with their own subcellular localisation and regulatory mechanisms has evolved, and these enzymes mediate the interconversion of glutamine, proline, and arginine 34,81 .A crucial step in the production of proline, ornithine, and arginine is the catalysis of glutamate by P5cS to produce P5c 64 .P5c is the precursor of proline; therefore, after a significant loop, this process returns to the beginning, i.e. proline.the "glutamine-arginine-proline metabolic axis" is thus formed by these metabolic pathways 79 .
the glutamine-arginine-proline axis is a crucial aspect of cancer metabolism and controls a significant proportion of amino acid metabolism.the production of other nonessential amino acids and necessary metabolites also relies on this axis as a framework 79 .Under various nutritional stress conditions, glutamine, arginine, and proline interconversions are significant 34 .Although some studies in adult humans have shown that enteral glutamine provides approximately 50% of the available carbon for arginine synthesis and that proline provides approximately 40% of the available carbon 82,83 , other research groups have shown that glutamine contributes more substantially to endogenous arginine synthesis in humans 63 . in addition, the formation of alpha KG from glutamate derived from either proline or arginine may play an anaplerotic role in the tcA cycle 34 .P5c acts as a carrier or transfer mechanism for the pyridine nucleotide-dependent oxidising, regardless of whether it is generated from arginine by ornithine, glutamate by P5cS, or proline by POX/PRODH 34 .in addition, the production of polyamines is derived from arginine 79 .An indication that glutamine may be converted to arginine for polyamine production is that the beneficial effects of glutamine deprivation on t-cell activation are partially reversed by polyamine supplementation 84 .these findings reinforce the already significant role of glutamine in cancer growth and proliferation and highlight the importance of the glutamine-arginine-proline metabolic pathway in cancer progression and treatment.

Advances in cancer therapy targeting the glutaminearginine-proline metabolism axis
the important role of the glutamine-arginine-proline metabolic pathway in the growth of cancer cells provides opportunities for cancer therapy targeting metabolism.to date, several studies have investigated the targeting of important proteins and enzymes in the glutamine, arginine and proline metabolic pathways to interfere with glutamine-arginine-proline metabolism 85 .(1) Glutamine metabolism: inhibitors such as glutamine transporters, GlS and transaminase have shown certain effects in preclinical and clinical treatment trials in patients at different stages of cancer.the current strategy for developing inhibitors is based on maintaining the level of glutamine metabolism needed for normal tissue physiology while impairing glutamine addiction in cancer cells 86 .the first step is to inhibit glutamine uptake, which can be depleted from the extracellular space by using l-asparaginase 31,87 .Alternatively, glutamine transport can be dampened by inhibiting ASct2 uptake via GPNA and v-9302.Second, the conversion of glutamine to glutamate can be inhibited by inhibiting GlS. the relevant inhibitors include DON, azaserine, acivicin, PteS, cB-839, and the small-molecule inhibitor 968, which can block GlS activity and glutaminolysis.Among these agents, DON, azaserine, and acivicin have been discontinued as early antagonists of glutamine metabolites due to excessive side effects, such as dose-limiting neurotoxicity, gastrointestinal toxicity (nausea and vomiting), and bone marrow suppression, even though they all exerted potent antitumour effects and exhibited clinical activity 87 . in light of the clinical potential of DON, several DON prodrugs (such as JHU-083 and DRP-104) have been developed to effectively decrease gastrointestinal toxicity 88 .JHU-083 is activated in vivo through the proteolytic hydrolysis of enriched enzymes (such as cathepsin-l), leading to the release of active DON.As a consequence, JHU-083 exhibits enhanced oral bioavailability and reduced toxicity 89 .the stability of DRP-104 in human plasma and gastrointestinal tissues, as well as the selective targeting of DON to tumours, have demonstrated its potential as an effective antitumour drug in various preclinical models without significant toxicity.consequently, this chemical has been chosen for clinical trials aimed at combating NSclc and other solid tumours 90 .BPteS and its derivative cB-839 inhibit the transition of GlS from dimer to tetramer, and the small-molecule inhibitor 968 inhibits the activity of Rho GtPases and thereby inactivates GlS 31 .Among these agents, cB-839 has entered phase ii clinical trials for the treatment of PiK3cA-mutant colorectal cancer (Nct02861300), NSclc (Nct03965845) and triple-negative breast cancer (Nct03057600). in addition, there are many novel GlS inhibitors.However, to date, only cB-839 has entered clinical trials for the treatment of advanced solid tumours and haematologic malignancies 91 .(2) Arginine metabolism: ASS1 is recognised as the rate-limiting enzyme in arginine synthesis, and in many cancers, including hepatocellular carcinoma, melanoma, prostate cancer, and renal cancer, ASS1 expression is downregulated or even absent, thus preventing cells from synthesising arginine and making them dependent on exogenous arginine.these cancers are thus sensitive to arginine deprivation therapies and are termed arginine-nutrient-deficient cancers 92 .Arginine deprivation is emerging as a promising new clinical treatment strategy for metabolism-based cancer therapy 93 .the antitumour activity of ADi-PeG20, an inhibitor targeting ADi, has been further demonstrated in pancreatic, prostate, small cell lung, and breast cancers 71 .Studies have shown that the antitumour activity of ADi-PeG20 is correlated with ASS1 deficiency in tumours, and its therapeutic effect may be eliminated when ASS1 is restored 70 .Phase iii clinical trials of ADi-PeG20 for the treatment of advanced or metastatic smooth muscle sarcoma (Nct05712694) and phase iii clinical trials for the treatment of patients with advanced liver cancer (Nct05712694) are currently underway.(3) Proline metabolism: the discovery of inhibitors targeting PYcR is at an early stage 94 .A remaining unresolved challenge is the high-resolution structure of human PRODH and PYcR.Screening for human PRODH inhibitors will be more challenging due to the lack of a convenient method for generating recombinant enzymes 33 .tanner et al. identified five small-molecule inhibitors of PYcR through a small-scale focused screening of proline analogues using X-ray crystallography.Of these, NelP was the most potent inhibitor and achieved its inhibitory effect by occupying the binding site of P5c 94,95 (table 1).
As connectivity Map (cMap) can be used to discover the mechanism of action of small molecules, functionally annotate genetic variants of disease genes and inform clinical trials, correlating the main data with that from other clinical databases would be fascinating 96,97 .we enriched the genes related to glutamine metabolism, arginine metabolism and proline metabolism by KeGG pathway analysis and used "Qurey" in the cMap online database to  .cmap analysis was performed to identify potential therapeutic drugs that target glutamine, arginine and proline metabolism.(a) cmap analysis was performed to identify potential therapeutic drugs that target glutamine metabolism.(b) cmap analysis was performed to identify potential therapeutic drugs that target arginine metabolism.(c) cmap analysis was performed to identify potential therapeutic drugs that target proline metabolism.Potential therapeutic drugs were analysed using connectivity map (https://clue.io) 96.
obtain the information about interactions between genes and drugs.Unfortunately, the corresponding small molecules listed in table 1 were not found (Figure 3).As mentioned above, various strategies and small-molecule inhibitors have been successively developed to target all aspects of glutamine-arginine-proline metabolism in cancer cells.P5cS, encoded by AlDH18A1, is the key enzyme that catalyses the conversion of glutamate to proline, ornithine and arginine 65 .changes in AlDH18A1 undoubtedly cause alterations in the glutamate-proline-arginine metabolic system, and these alterations affect the processes of tumorigenesis and progression.therefore, a further review of the clinicopathological characteristics, evolution, clinical significance, and drug development prospects of AlDH18A1 and cancers is provided below 98 .

ALDH18A1 protein structure
AlDH18A1 is located on chromosome 10 (10q24.3),contains 19 exons and is approximately 50 kb in length.P5cS contains two enzymatic structural domains, γ-GK (amino acids 1 to 361) and γ-GPR (amino acids 362 to 795): the former contains 64 amino acid residues in the mitochondrial targeting sequence that mediate the phosphorylation of glutamate to form γ-GP, and the latter converts γ-GP, an unstable intermediate reaction product, into P5c and its isomer GSA. the crystal structure of γ-GPR has been resolved (PDB iD 2H5G) (Figure 4(A)), and the crystal structure of the GK portion of P5cS remains unresolved but was predicted by the AlphaFold protein structure database (Figure 4(B)) 99 .

Expression and significance of ALDH18A1 in cancers
As an important aldehyde dehydrogenase isoform AlDH18A1 exhibits the highest activity in the small intestine and is expressed in the colon, thymus and brain 65 .AlDH18A1 is involved in the generation of a variety of epigenetically regulated intermediates in cancer, such as α-KG 100 , NAD(P) +57 , and SAM 101 , and plays an important role in metabolic reprogramming and epigenetic regulation of the effects of interconnections in cancer.
in P493 and Pc9 cells, AlDH18A1 affects NAD + /NADH to regulate the glycolytic pathway and thereby regulates proliferation by altering cellular energy metabolism; furthermore, AlDH18A1 affects NADP + /NADPH to regulate the pentose phosphate pathway and proliferation via nucleotide metabolism 57 .P5c can be converted to proline in the mitochondria or cytoplasm via PYcR using NADPH or NADH as a cofactor for conversion to proline.thus, the interconversion of P5c and proline leads to the recirculation of cellular NAD(P)H to NAD(P) +102 .consistent with recent studies, Ding et al. also reported that AlDH18A1 can influence the glycolysis and pentose phosphate pathways in hepatocellular carcinoma cells through the production of ADP + , which is essential for nucleotide biosynthesis and the production of acetyl coenzyme A 103 .thus, AlDH18A1 promotes hepatocellular carcinoma cell proliferation, and inhibition of AlDH18A1 may be a novel therapeutic strategy for hepatocellular carcinoma 103 .
in addition, hypoxia or etc inhibition induces reductive glutamine metabolism, in which glutamine is converted primarily to acetyl coenzyme A for fatty acid biosynthesis 37 .cancer cells often survive under hypoxic conditions and undergo metabolic reprogramming.Furthermore, the knockdown or inhibition of AlDH18A1 in luminal B breast cancer may contribute to the therapeutic effects resulting from targeting this enzyme.Furthermore, high protein expression of glutamine-prolinases (GlS, AlDH18A1 and P5cR) is associated with high c-Myc protein expression in only luminal B breast cancer, suggesting that c-Myc is the driving force behind the metabolic state of the breast cancer subclass 104 .Proline biosynthesis is significantly enhanced in melanoma cells compared with normal melanocytes.the inhibition of AlDH18A1 can inhibit cancer growth by 60% to 99% and reduce the viability of is available for free consultation and download on the uniprot online platform (https://www.uniprot.org) 132.melanoma cells by 90%, whereas exogenous supplementation with proline can reverse this effect 105 . in addition, knockdown of MYc significantly reduces proline and hydroxyproline production in hepatocellular carcinoma cells under hypoxic conditions, indicating that the glutamine metabolic axis via MYc/GlS and MYc/ AlDH18A1 is essential for proline synthesis under hypoxic conditions 106 .Furthermore, Fang et al. reported that AlDH18A1 and PYcR1 are frequently overexpressed in NB cells and human cancers and are associated with poor patient prognosis, whereas the deletion of AlDH18A1 or PYcR1 decreases proline levels and reduces tumorigenesis 49,105 .Fang et al. reported that the expression levels of AlDH18A1 or PYcR1 are associated with poor prognosis in NB patients 107 .consistent with the results of this study, our previous study also demonstrated that the expression of AlDH18A1 was significantly associated with overall survival of NB patients.Administration of the AlDH18A1-specific inhibitor YG1702 inhibits N-MYc expression and attenuates NB cell growth 108 . in conclusion, AlDH18A1 may be a potential drug target for cancer therapy (table 2). to further verify the expression of AlDH18A1 in different tumour cell lines, we downloaded cell line data from the Human Protein Atlas (HPA) database 109 .the HPA was used to summarise the transcriptional level of each gene in 1206 cell lines.the analysed cell lines were grouped according to cancer type (Figure 5(A)). in addition, we further verified the expression of AlDH18A1 mRNA in different tumour tissues and normal tissues using the Gene expression Profiling interactive Analysis (GePiA) online database 110,111 , and the results showed that the expression of AlDH18A1 was upregulated in 16 types of tumour tissues compared to that in normal tissues (Figure 5(B)).

Anticancer therapy targeting ALDH18A1
AlDH18A1 is highly expressed in a range of tumours, including NB 108 , hepatocellular carcinoma 103 and luminal B breast cancer 104 .Because it is a crucial enzyme in the glutamine-arginine-proline metabolic system, targeting AlDH18A1 via anticancer therapies could create novel opportunities for cancer intervention.to identify specific small-molecule inhibitors of AlDH18A1, structure-based computerised virtual screening, which is a very effective method for high-throughput screening of drugs, was utilised.Structure-based virtual screening includes various sequential computational stages, target and database preparation, docking and postdocking analysis, and prioritisation of test compounds 112 .Among these steps, conformational changes due to interactions with ligands and structural resolution are important details to be considered when selecting the most suitable structure.However, the current algorithms cannot estimate the absolute energy associated with intermolecular interactions with satisfactory accuracy.the appropriate evaluation of solvent effects, entropic effects and receptor flexibility are the main challenges that need attention.
the P5cS protein encoded by AlDH18A1 is known to contain two enzymatic structures, the G5PR and G5K structural domains.the crystal structure of the G5K portion of P5cS has not been resolved.therefore, we used molecular docking to screen for P5cS-specific small-molecule inhibitors based on the structure of G5PR and ultimately selected the compound with the highest docking score to P5cS, namely, YG1702 108 .Furthermore, we verified the specific binding between YG1702 and the AlDH18A1 recombinant protein using isothermal titration calorimetry and found that YG1702 has a high affinity for AlDH18A1 and may affect its enzymatic activity 108 .Because the partial structure of G5K, another enzymatically active structural domain of human P5cS, has not been resolved, screening of inhibitors with enzymatic activity of this structural domain is difficult.However, the crystal model of the catalytic structural domain of Escherichia coli G5K has been successfully resolved, and AlDH18A1 is highly conserved in a variety of organisms 113 .Proteins with relatively high sequence and functional conservation are more desirable target molecules for drug design.if there is a mutation of a single amino acid residue within the active site of the protein (point mutation) that may affect ligand binding, the drug molecule targeting the mutated protein will not exhibit the desired effect due to the high likelihood of mutations in the sequence of the structural domain 114 . in addition, an important issue in molecular docking is finding the best binding site between the substrate and the target enzyme.Due to the complexity and variability of the tertiary structure of proteins, identifying a suitable region where the ligand can bind easily is the basis of molecular docking methods.the results of molecular docking are extremely sensitive to protein conformation 115 .therefore, the structural conservation of proteins is highly convenient for drug development 116 .
in addition, there is a need to investigate the possible side effects of targeted therapy for AlDH18A1, which is a key enzyme involved in the glutamate-arginine-proline metabolic system and therefore essential for normal tissue function.Defects in AlDH18A1 may lead to congenital disorders such as hypoprolinemia, hypoarginemia, neurodegeneration, and connective tissue  (b) transcript expression of aldh18a1 in clinical tumour samples and normal tissues in the GEPIa database.the red box plots show tumour expression, while the grey colours represent normal tissues.aldh18a1 mrna expression was analysed using the free public databases human Protein atlas (hPa, http://www.proteinatlas.org) 133 and Gene Expression Profiling Interactive analysis (GEPIa, http://gepia.cancer-pku.cn/) 110.
abnormalities 117 .Determining whether these phenotypes occur when targeting AlDH18A1 is very important, and the possible side effects can be mitigated when cancer treatments are targeted.in addition, drugs targeting AlDH18A1 should not disrupt the function of other AlDH family members that may be necessary for normal physiological processes 105 , such as AlDH1As, an important subfamily of AlDH that includes 3 members: AlDH1A1, AlDH1A2 and AlDH1A3.AlDH1As and AlDH8A1 jointly catalyse the second step of RA synthesis, namely, the oxidation of all-trans or cis-retinal to RA, and are important regulatory genes involved in RA synthesis 118 ; RA synthesis is essential for the normal growth, development and maintenance of adult vertebrate epithelial cells 119 .AlDH2, as a nitrate reductase, is the main enzyme necessary to activate nitroglycerine and used to treat angina and heart failure 120 .AlDH3A1 is considered a corneal crystallin, is one of the most abundantly expressed proteins in mammalian corneal epithelial cells, and accounts for more than 50% of the total water-soluble protein fraction, and corneal AlDH3A1 protects the cornea and underlying lens from Uv-induced oxidative stress 121 .

Prospects
Metabolic abnormalities are one of the hallmarks of cancer.Metabolic reprogramming promotes cancer cell survival, proliferation, angiogenesis and metastasis.An increasing number of in-depth studies on cancer metabolism have shown a strong link between amino acid metabolism and cancer behaviour 122 .Glutamine is the most abundant amino acid in plasma, is used to replenish the tcA cycle and provides carbon and nitrogen for the synthesis of nucleotides, amino acids and glutathione.Notably, glutamine, arginine and proline metabolism play important role in epigenetic regulation by altering the expression of tumour oncogenes and tumour suppressor genes and the cancer cell phenotype 123,124 .considering the broad opportunities for targeted metabolic therapies for cancer, a key question is whether researchers have exhausted all metabolic hubs as targets, despite the successive development of several strategies and small-molecule inhibitors targeting various aspects of glutamine-arginine-proline metabolism in tumour cells 31 .in particular, changes in AlDH18A1, a key enzyme in the glutaminearginine-proline metabolic system, undoubtedly affect the processes of tumorigenesis and progression 105 .AlDH18A1 not only regulates proline metabolism in cancer cells such as melanoma, NB and hepatocellular carcinoma cells but also controls the pentose phosphate pathway by altering the NADP + /NADPH ratio, which in turn regulates cell proliferation through nucleotide metabolism in cancer cells such as lymphoma, NSclc and hepatocellular carcinoma cells 57,103 .therefore, further exploration of the function of AlDH18A1 in cancers has become increasingly relevant.we previously used a molecular docking approach to screen for specific AlDH18A1 inhibitors.Among these compounds, YG1702 exhibited low cytotoxicity in normal human brain glial (HeB) cells and human colonic mucosal epithelial NcM460 cells but high cytotoxicity in MYcN-amplified NB cells.isothermal titration calorimetry (itc) showed high affinity between AlDH18A1 and YG1702 with a low Ka.YG1702 not only inhibits AlDH18A1 expression but also leads to the downregulation of MYcN expression, which provides a possible indirect strategy for MYcN targeting.the in vivo results obtained for YG1702 have revealed significant inhibition of NB xenografts in animal models of cancer.However, our experiments did not validate the conformational relationship or chemical stability of YG1702, which will be required before its use in vivo.More reliable experimental validation of the chemical structure of YG1702, such as in vitro testing of YG1702 analogues, is necessary to demonstrate that this compound is indeed a lead drug candidate.the in vitro inhibitory activity of this compound against AlDH18A1 should also be quantitatively demonstrated.YG1702 has two ester groups, which typically exhibit low stability and are frequently hydrolysed.Although in vitro and in vivo experiments have demonstrated the effectiveness of YG1702, some issues remain that need to be further addressed.(1) the solubility of YG1702 is poor and should be increased by cyclodextrin inclusion, microemulsion or liposomes, and micelles.(2) the inhibitory effect of YG1702 on AlDH18A1 enzyme activity needs to be further investigated.(3) the structure of YG1702 should be explored to improve the stability and effectiveness of the inhibitor.
Addressing the following unknowns will help us more fully understand the role of AlDH18A1 in cancers and develop more effective therapeutic measures: (1) as mentioned above, P5cS.long and P5cS.short, which are obtained by exon sliding of the AlDH18A1 gene, are distributed in different tissues.P5cS.long is mainly involved in glutamate biosynthesis, and P5cS.short is involved in the biosynthesis of arginine, which suggests that these two forms of P5cS play different roles in different cancer environments.therefore, determining whether the expression and function of these two forms of P5cS shift to adapt to the cancer microenvironment is of interest.(2) Because AlDH18A1 is a potential target in cancer therapy, its specific inhibitor must be explored.the crystal structure of P5cS has been deposited in the RcSB protein database (PDB iD: 2H5G).therefore, molecular docking can be used to develop targeted P5cS inhibitors or ligands; this approach is common in drug design.However, P5cS contains two enzyme structural domains, γ-GK and γ-GPR.which structural domain is more suitable for designing specific inhibitors remains unclear, and specific compounds based on γ-GK and γ-GPR may exhibit different inhibition efficiencies.(3) Glutamine is transported into the cell by AcSt2 and then converted to glutamate by GlS. in addition, glutamate is converted to α-KG, which participates in the tcA cycle or is converted to proline or arginine by AlDH18A1.when the expression or activity of AlDH18A1 is altered, other glutamate-related metabolic enzymes in cancer cells may be regulated to compensate for this effect.therefore, the relationship between AlDH18A1 and other glutamate-related metabolic enzymes must be investigated.(4) cScs, also known as tics, are a small subpopulation of cancer cells that promote tumorigenesis, progression, drug resistance, and recurrence.there is clear evidence showing that some members of the AlDH family, particularly AlDH1A3 and AlDH1A1, are expressed in cScs and can regulate cellular functions 125 .various AlDHs play specific biological roles in cancer cells.Because AlDH18A1 is an enzyme involved in amino acid metabolism, its expression level and function in cScs are unknown and need to be further investigated.in conclusion, understanding the regulatory role of AlDH18A1 in tumorigenesis, progression and drug resistance would be helpful.

Figure 1 .
Figure1.functions of P5cs in different tissues.mammalian P5cs undergoes alternative splicing to produce two isoforms.P5cs.short (the short isoform), which is inhibited by ornithine and converts glutamine to P5c, has high activity in the intestine.P5cs.long (the long isoform) has a broad tissue distribution, is insensitive to ornithine inhibition, and is involved in proline biosynthesis.this pathway is regulated by proline inhibition of P5cr.abbreviations: P5c: pyrroline-5-carboxylic acid; P5cs: pyrroline-5-carboxylic acid synthase; P5cr: pyrroline-5-carboxylic acid reductase.the elements of the figure were downloaded for free from biorender.comand then combined and drawn by the author.

Figure 2 .
Figure 2. Interactions among glutamine-arginine-proline metabolism.P5c acts as a carbon skeleton and is an important intermediate metabolite in the interconversion of glutamine, arginine and proline.Gsa and P5c are tautomers.their interconversions are spontaneous.the sources of glutamine are shown on the top left, and the source of proline is shown on the top right.Glutamine enters the cell via slc1a5 and can be converted to glutamate by glutaminase.P5cs is the first enzyme that converts glutamate to proline.Proline is converted to arginine via the P5c intermediate.arginase then catalyses the forward conversion of arginine to ornithine, and oat reversibly converts ornithine to P5c. activation of the oncogenes K-ras and myc further increases slc1a5 expression.myc increases the gene expression of P5cs and Pycr, thereby increasing glutamine and proline uptake and metabolism rates.abbreviations: Ecm: extracellular matrix; Gsa: gamma-glutamyl semialdehyde; P5c: pyrroline 5-carboxylate; Glu: glutamate; Gln: glutamine; Pro: proline; Gls: glutaminase; P5cs: P5c synthase; P5cdh: P5c dehydrogenase; P5cr: P5c reductase; Prodh: proline dehydrogenase; oat: ornithine-δ-transaminase; orn: ornithine; Pa: polyamine; otc: ornithine transcarbamylase; ass: argininosuccinate synthase; asl: argininosuccinate lyase; arG: arginine; arGases: arginase enzymes, including arG1 and arG2.the elements of the figure were downloaded for free from biorender.com and then combined and drawn by the author.

Figure 3
Figure3.cmap analysis was performed to identify potential therapeutic drugs that target glutamine, arginine and proline metabolism.(a) cmap analysis was performed to identify potential therapeutic drugs that target glutamine metabolism.(b) cmap analysis was performed to identify potential therapeutic drugs that target arginine metabolism.(c) cmap analysis was performed to identify potential therapeutic drugs that target proline metabolism.Potential therapeutic drugs were analysed using connectivity map (https://clue.io)96 .

Figure 4 .
Figure 4. crystal structure of human P5cs.(a) crystal structure of human P5cs (Pdb Id 2h5G) at amino acid positions 362-795 from the Pdb database.(b) Predicted crystal structure of human P5cs (af-P54886-f1) at amino acid positions 1-795 from the alphafold protein structure database.the crystal structure of human P5cs is available for free consultation and download on the uniprot online platform (https://www.uniprot.org) 132.

Figure 5 .
Figure 5. aldh18a1 mrna expression in patients with various types of cancer.(a) aldh18a1 mrna expression data are expressed as normalised transcript per million (ntPm) values in 1206 cancer cell lines from the human Protein atlas (hPa) database.the data are based on hPa version 23.0 and Ensembl version 109.(b) transcript expression of aldh18a1 in clinical tumour samples and normal tissues in the GEPIa database.the red box plots show tumour expression, while the grey colours represent normal tissues.aldh18a1 mrna expression was analysed using the free public databases human Protein atlas (hPa, http://www.proteinatlas.org)133 and Gene Expression Profiling Interactive analysis (GEPIa, http://gepia.cancer-pku.cn/)110 .

Table 1 .
Pharmacological strategies targeting the glutamine-arginine-proline metabolic system in cancer.

Table 2 .
aldh18a1 is highly expressed in diverse types of cancer.